Identification of Genes Involved in Wild Crucifer <i>Rorippa indica</i> Resistance Response on Mustard Aphid <i>Lipaphis erysimi</i> Challenge

Mustard aphid, Lipaphis erysimi (L.) Kaltenbach is a perpetual annual threat in the cultivation of rapeseed- mustard (Brassica spp.) crop in tropical and sub-tropical climate. Cultivated Brassica germplasm has failed so far to provide any source of resistance. Wild germplasm is a potential source of resistance against many threatening herbivores. On wild germplasm screening, we noted that the wild crucifer Rorippa indica (L.) Hiern confers resistance against L. erysimi. In the present study L. erysimi challenged transcriptome of R. indica was compared to un-infested R. indica sample to get a molecular insight about the aphid resistance mechanism and identify the candidate defense response genes. Cloning, sequencing and in silico sequence analysis of complimentary DNA amplified fragment length polymorphism identified 116 differentially expressed transcript derived fragments revealed thirty candidates which are from different functional categories including redox regulation, signalling, photosynthesis, structure, metabolism, defense response as well as a few of unknown function. Twenty four identifications were then studied by quantitative real time RT PCR analysis at 6, 12, 24 and 48 hour time point post infestation to understand the early-to-late defense response through their relative gene expression profiles. Seventeen fragments showed significant up or down regulation at p<0.05 level. The response was influenced by different phytohormonal signalling pathways simultaneously. The candidate defense response expressed sequence tags specifically for the resistance genes identified in this study have implication in building desired mustard aphid resistance in susceptible rapeseed-mustard plants in future. This is the first molecular report on crucifer defense response against mustard aphid L. erysimi.芥菜蚜（Lipaphis erysimi (L.) Kaltenbach）是热带、亚热带气候区油菜-芥菜类作物（Brassica spp.）种植中常年持续存在的虫害威胁。截至目前，栽培芸苔属种质资源尚未筛选到任何有效的抗蚜抗源。野生种质资源则是抵御多种重大植食性虫害的潜在抗源库。通过对野生种质资源的系统性筛选，我们发现野生十字花科植物印度蔊菜（Rorippa indica (L.) Hiern）对芥菜蚜具有稳定抗性。本研究以受芥菜蚜侵染的印度蔊菜转录组与未接虫的对照组样本进行对比分析，旨在从分子层面解析其抗蚜机制，并筛选候选防御响应基因。通过对互补DNA扩增片段长度多态性（cDNA-AFLP）产物的克隆、测序及生物信息学序列分析，我们在116个差异表达转录衍生片段中鉴定出30个候选基因，这些基因涵盖氧化还原调控、信号转导、光合作用、结构组成、代谢过程、防御响应等多个功能类别，另有部分基因功能尚未明确。随后，我们选取其中24个候选基因，在接虫后6、12、24及48小时四个时间点开展实时荧光定量逆转录聚合酶链反应（qRT-PCR）分析，通过相对基因表达谱解析其从早期到晚期的防御响应动态过程。结果显示，17个片段在p<0.05的显著性水平下呈现显著上调或下调表达。该防御响应过程同时受到多种植物激素信号通路的协同调控。本研究鉴定得到的抗蚜候选防御响应表达序列标签，未来可应用于培育具备稳定芥菜蚜抗性的易感型油菜-芥菜类作物。本研究亦是全球范围内首个针对十字花科植物抵御芥菜蚜（L. erysimi）的分子层面研究报告。